Method of preparing 16-oxygenated derivatives of estr-4-en-3-one



United States Patent C) 3,329,579 METHOD OF PREPARING 16-OXYGENATEDDERIVATIVES OF ESTR-4-EN-3-0NE Karl Jolivette Sax, West Nyack, andRobert Henry Blank and Chester Eric Holmlund, Pearl River, N.Y., andRalph Henry Evans, Jr., River Vale, N.J., assignors to American CyanamidCompany, Stamford, Conn., a corporation of Maine No Drawing. Originalapplication Jan. 24, 1964, Ser. No. 339,851, new Patent No. 3,226,404,dated Dec. 28, 1965. Divided and this application Oct. 5, 1965, Ser. No.493,261

4 Claims. (Cl. 19551) ABSTRACT OF THE DISCLOSURE Preparation of16,8-hydroxy-estr-4-en-3-one and estr- 4-ene-3,l6-dione by contactingCephalosporium acremonium with estr-4-en-3-one under fermentationconditions, and the preparation of 16-hydroxy or 16-ketoestr-4-en- 3-oneby contacting a growing culture of Bacillus megaterz'um withestr-4-en-3-one. The compounds are useful as hypocholestermic agents andin some instances, for their anabolic activity.

This application is a division of application Ser. No. 339,851, filedJan. 24, 1964, now US. Patent 3,226,404.

The novel process of the present invention is essentially amicrobiological oxidation of steroid substrates to produce the16-oxygenated derivatives. The type of oxygenated steroid depends on theorganism selected and the particular fermentation conditions.

The starting material may be for example, 3-methoxyestra-1,3,5()-trienewhich is chemically converted to 3-methoxyestra-2,5(10)-diene bytreatment with lithium and liquid ammonia. The3-methoxyestra-2,5(10)-diene on treatment with hydrochloric acidproduces estr-4-en- 3-one, the substrate used in the fermentationsdescribed hereinafter to produce the 16-oxygenated estr-4-en-3-ones.

In carrying out the process of the present invention, the suitableorganism is cultivated aerobically in a suitable nutrient medium withthe steroid which is to be hydroxylated. During the growth of theorganism under favorable conditions, the desired hydroxyl group isintroduced into the steroid ring structure. The exact mechanism by whichthis hydroxylation is accomplished is not wholly certain. It is known tobe caused by enzymes produced by the organism in the process of growth.

In accordance with the present invention, suitable organisms of thegenus Bacillus such as Bacillus megaterium, or of the genusCephalosporium such as Cephalosporium acremonium have the unexpectedproperty of producing the desired result. These organisms are on depositat culture depositories such as Northern Regional Research Laboratories,Peoria, Ill. and as described hereinafter.

A suitable nutrient medium contains a soluble source of carbon, nitrogenand mineral elements. In general, preparation of such media is wellknown and the practice of the present invention in this respect mayfollow such procedures. An illustrative example of a suitable medium ofthis type is one which contains the following: 2% Edamin, 2% glucose,0.5% cornsteep liquor; the whole being adjusted to about pH 7 with10-normal sodium hydroxide. This medium is used in the illustrativeexamples below.

In an illustrative preparation of inoculum, about one ml. of cellsuspension as washed with about six ml. of aqueous sterile salt solution(0.9%) from a suitable culture, as on potato dextrose agar, yeastextract agar or the like, is used to inoculate about 100 ml. of sterilemedium in ice a 500 ml. Erlenmeyer flask. Inoculated medium then isincubated at about 25 -37 C., usually averaging about- 28 C., on ashaker for an initial pregrowth period of some 4 to 24 hours. Thereafterthe steroid is added, usually about 10 mg. in about 1 ml. of methanol,and incubation is continued until harvest, usually some 24-144 hoursafter adding the steroid. This procedure is used below in theillustrative examples.

A number of alternatives for the several steps may be used, for example,use of a pregrown vegetative inoculum instead of the washed slantsuspension. Other media, media volumes, incubation periods, andtemperatures may be used. Instead of adding the steriod to the inoculathe latter may be used to inoculate other flasks for steroid conversionor larger batches of medium in seed bottles. Such bottle cultures, afterfurther incubation, usually under conditions of aeration and stirringmay be used for steroid conversion or to inoculate large batches ofmedium in aerated, stirred fermentation tanks.

A good typical practice in fermentation tank procedure is illustrated inthe following method. Medium is prepared in the tank, sterilized byheating and cooled to the temperature of the inoculant. The medium isthen inoculated with from about one to about six percent of a vegetativeinoculum prepared as above. The broth is then agitated with a stirrer atfrom about to about 500 r.p.m., and aerated at the rate of about 0.5 toabout 1.5 volumes of air per volume of broth per minute. The actualvalues used will vary within the approximate ranges shown, dependingupon the volume, the shape of the tank, the stirrer and the like.

The inoculated tank is then maintained at the desired temperature, inthe illustrative case about 27 C., for from 6 to 70 hours. The steroidsubstrate is then added, usually dissolved in methanol. Agitation andaeration are continued until the substrate has been substantiallyconverted to the desired products. The culture broth is then harvestedand the hydroxylated product isolated.

The amount of steroid added as substrate to the fermentation may bevaried as necessary or desirable. However, a good practice willordinarily be to add on the order of about 0.05 to about 1.0 gram perliter of nutrient medium.

When using such steroid substrates in the fermentation, the productsformed are the free steroids. The steroid substrates are generally addedto the fermentation in solution or in finely-divided form. A preferredmethod is to dissolve the steroid in methanol or some otherwatermiscible solvent and to add it to the fermentation medium at thedesired stage in the process. Although the steroid may precipitate fromsolution when so added, it is dispersed throughout the medium as a finesuspension and becomes readily available to the organism for oxidation.

During fermentation, it is frequently found desirable to add anantifoaming agent. In such cases, commerciallyavailable products may beused. These usually contain such agents as silicones, glyceride oils,and the like.

The compounds of this invention are useful as intermediates for themicrobial dehydrogenation process which yields 16-oxygenated17-desoxoestrones, which are useful as hypocholesteremic agents. Anadditional utility of the invention is illustrated by compounds of thisinvention, such as l6fi-hydroxyestr-4-en-3-one which demonstrates ahighly desirable ratio of anabolic to androgenic activity. That is tosay, while the compound has a high anabolic activity, at the same timeit shows low androgenic activity.

The invention broadly described will be further illustrated in greaterdetail by the following specific examples. Preparation of the initialfermentation substrate, estr-4-en- 3-one, is described in Examples 1 and2. It should be understood however, that although these examples maydescribe in particular detail some of the more specific features of thepresent invention, they are given primarily for the purpose ofillustration, and the invention in its broader aspect is not to beconstrued as limited thereto.

EXAMPLE 1 Preparation of 3 -methxyestra-2,5 I 0 -diene A partialsolution of 7.5 g. of 3-methoxyestra-1,3,5 (10)-triene in 20 ml; ofetthanol, 100 ml. of anhydrous ethyl ether, and 500 ml. of liquidammonia is stirred and 7.0 g. of lithium, cut into small pieces, isadded during two hours. Additional liquid ammonia is added as necessaryto maintain the volume. The reaction is continued until an 0.2 ml.aliquot diluted to 10 ml. with ethanol no longer shows appreciable ultraviolet absorption at 280 mg. The reaction mixture is warmed to evaporatethe ammonia, and the excess lithium is destroyed by the careful additionof ethanol and water. After dilution with water the mixture is extractedseveral times with ether and the combined ether extract is washed withwater, dried over magnesium sulfate, and filtered. Concentration of thefiltrate and fractional crystallization of the product from other yields4.82 g. of 3-methoxyestra-2,5 (10)-diene, melting point 91-93", +69(chloroform). Additional product may be isolated from the motherliquors.

EXAMPLE 2 Preparation of estr-4-en-3-0nc A suspension of 5.72 g. of3-methoxyestra-2,5(10)- diene in 48 ml. of methanol and 2 ml. ofconcentrated hydrochloric acid is warmed gently on a steam bath untilsolution is complete (about minutes). The reaction mixture is allowed tostand for minutes, and is then treated with 5 ml. of 5 N sodiumhydroxide solution and a little water. After cooling in a mixture ofsolid carbon dioxide with methanol the solution deposits 4.2 g. ofcrystalline estr-4-en-3-one, melting point 63.4-65 C. Recrystalliza tionof this material yields pure estr-4-en-3-one, melting point 65.566.75C.,

iggg 240 m 616,000, M+44 (chloroform), 0.1%

+24 (methanol) EXAMPLE 3 Preparation of 16/3-hydr0xyestr-4-en-3-0ne Aninoculum containing Bacillus megateriam (NRRL- B938) is added understerile conditions to a fermentor containing a sterile growth mediumconsisting of 1500 g. of glucose, 600 g. of Edamin, and 150 g. ofcornsteep liquor in sufficient water to make 30 liters and adjusted topH 6.5 with sodium hydroxide. The fermentation mixture is stirred andaerated at 28 C. during 48 hours, at which time a solution of 3.0 g. ofestr-4-en-3-one in methanol is added. Six and one half hours after theaddition of steroid the fermentation mixture is adjusted to pH 6.5 withhydrochloric acid, and extracted with an equal volume of chloroform. Thechloroform layer is separated and the aqueous layer is twice extractedwith half its volume of chloroform.

The combined chloroform extract is concentrated under reduced pressureto 4 liters, dried over magnesium sulfate, and after stirring forseveral minutes with 5 g. of decolorizing carbon, is filtered.Concentration of the chloroform yields an oily residue, which isdissolved in 200 ml. of 75:25 (volume) methylene chloride-hexanemixture. Adsorption chromatography on a 300 g. silica gel column isaccomplished by elution with 1800 ml. of the above solvent followed by 8liters of 2% acetone-98% methylene chloride and 3 liters of 10% acetonein methylene chloride. The steroid obtained by evaporation of solventfrom the 3800-6800 ml. portion of the eluate is crystallized fromacetone-hexane to yield 500 mg. of 16,8-hydroxyi estr-4-en-3-one,melting point 149l50 C., [ch +22.8 (c.=1.05 in CH OH),

EXAMPLE 4 Preparation 0 f estr-4-en-3,16-di0ne, 1 6 a-hydroxyestrend-oneand 1 6fl-hydr0xyestr-4-en-3-0ne An inoculum containing Bacillusmegaterium (NRRL B938) is added under sterile conditions to afermentation tank containing 400 g. of Edamin, 400 g. of glucose, and144 g. of cornsteep liquor in sufficient water to make 20 liters. Thefermentor contents are stirred and aerated at 28 C. for 23 hours. Twograms of estr-4-en-3-one is added in methanol solution, and, after 70hours of fermentation the contents of the fermentor are removed andextracted with an equal volume of chloroform after acidification to pH6.5 with hydrochloric acid. The chloroform layer is separated and theaqueous layer is twice extracted with half volumes of chloroform.

Evaporation of the chloroform from the combined extracts leaves 133 g.of a viscous oil, which is fractionated by adsorption chromatography ona column composed of 600 g. of silica gel. The column is developed with50% methylene chloride50% hexane (1.2 liters), 60% methylene chloride40%hexane (2 liters), methylene chloride-20% hexane (2 liters), methylenechloride (7 liters), 5% ether-% methylene chloride (1 liter), 10% ether90% methylene chloride (1 liter), 50% ether-50% methylene chloride (4liters) and ether (5 liters).

Partition chromatography of the residue obtained on evaporation ofsolvent from the 80% methylene chloridehexane and the first part of themethylene chloride eluate is done on a 400 g. column of diatomaceousearth moistened with 200 ml. of the lower phase of a solvent systemcomposed of water, methanol, dioxane and cyclohexane in the volume ratio2:4: 1 10. Elution of the column with 1 liter of the upper phase of theabove system and two liters of the upper phase of a similar system whoseproportions are 2:611:10 provides a fraction between 1035 and 1895 ml.of eluate, which yields, on concentration and crystallization of theresidue from aqueous methanol, 700 mg. of estr-4-ene-3,16-dione, meltingpoint 139.5"- 140.5 C. Additional less pure material is found in themother liquors.

The next portion of the methylene chloride eluate from the adsorptioncolumn is concentrated, and the residue is chromatographed on apartition column of 75 g. of diatornaceous earth moistened with 37.5 ml.of the lower phase of a solvent system composed of water, methanol,dioxane, and cyclohexane in the volume ratio 1:1:135. Elution of thecolumn with the upper phase of the solvent system shows concentrationsof steroids at 90l65 ml. and 2l5330 ml. of eluate. Concentration of thefirst fraction and crystallization of the residue from aqueous methanolyields 70 mg. of estr-4-ene-3,16-dione. Similar work up of the secondfraction provides 13 mg. of 165-hydroxyestr-4-en-3-one, melting point146l47.5 C.

The first 1270 ml. of the 50% ether-50% methylene chloride eluate fromthe adsorption column is concentrated to a residue which is furtherpurified by partition chromatography on a column of 300 g. ofdiatomaceous earth moistened with 150 ml. of the lower phase of asolvent system composed of water, methanol, dioxane, and cyclohexane inthe volume ratio 121:2:5. Elution with the upper phase of this systemyields the major component between 825 and 1265 ml. Crystallization fromethyl acetate-hexane of the residue obtained on concentration of thisfraction provides 157 mg. of 16a-hydroxyestr-4-en-3- one, melting point163164 C.,

EXAMPLE 5 Preparation of estr-4-ene-3,16-dione and 16,8-hydroxyestr-4-en-3-one An agar slant supporting an active growth of Cephalosporinmacremonium (NRRL No. 3092; Lederle culture Z918) is scraped understerile distilled water, and the suspension is used to inoculate 100 ml.of medium (consisting of 2% Edamin, 2% glucose and 0.5% cornsteepliquor) in a 500 ml. Erlenmeyer flask. The flask is shaken on a rotaryshaker at 28 C. for 64 hours and its contents are used to inoculatethree 750 ml. portions of the same medium in 4 liter Erlenmeyer flasks.The culture is shaken for 64 hours at 28 C., and a solution of 500 mg.of estr-4-en-3-one in a small amount of methanol is divided equallyamong the three fermentation flasks. After shaking at 28 C. for 51hours, the fermentations are combined and the pH is reduced from 6.9 to2.5 with 5 N hydrochloric acid.

The fermentation mixture is extracted twice with an. equal volume ofmethylene chloride. After filtration the filtrate is again extractedwith methylene chloride, and the combined extract is concentrated to 8.5g. of an oily residue. Partition chromatography of the residue on a 150g. column of diatomaceous earth moistened with 75 ml. of the lower phaseof a solvent system composed of water, methanol, dioxane, andcyclohexane (volume ratio 2:411:10) separates two steroid products. Oneis estr-4-en-3,l6dione, identified by mixture melting point with a knownsample and by infrared absorption spectrum. The other is16I3-hydroxyestr-4-en-3-one, melting point 146.5-148.5 C.

EXAMPLE 6 Preparation of 3-hydr0xyestra-1,3,5 (Z0)-trien-Z 6-one Three500 ml. Erlenmeyer flasks, each containing 100 ml. of medium #13consisting of cerelose 10 g., yeast extract, 1 g., 2.5 g. sodiumchloride, 4 g. beef extract, 4 g. peptone, and 1000 ml. of water,inoculated with the growth of Nocardia corallina (ATCC 999) from yeastextract agar slant, are shaken for 6 hours at 37 C. on a reciprocatingshaker. The contents of these flasks are used to inoculate three 750 ml.portions of fresh medium #13 contained in three 4 liter Erlenmeyerflasks. After incubation of this mixture on a reciprocating shaker at 24C. for 16 hours, 165 mg. of estr-4-ene-3,l6-dione is added to each flaskunder sterile conditions, and the fermentation is continued for 24hours.

The combined fermentation mixture is stirred with an equal volume ofmethylene chloride. After settling, the methylene chloride layer isseparated, and the aqueous layer is reextracted with two half volumes ofmethylene chloride. Evaporation of the methylene chloride from thecombined extracts provides an oily residue which is purified bypartition chromatography on a column composed of 200 g. of diatomaceousearth moistened with 100 ml. of the lower layer of a solvent systemcomposed of water, methanol, dioxane, and cyclohexane in the volumeratios 2:4:1z10. Elution of the column with the upper phase of the abovesolvent system (900 ml.) and the upper phases of systems containing thesame solvents in the volume ratios 2:211:10 (900 ml.) and 2:22:10 (1000ml.) separates the steroid from its accompanying impurities so thatcrystallization of the residue obtained on evaporation of solvent fromthe eluate provides a 67% yield of3-hydroxyestra-1,3,5(10)-trier1-16-one, melting point 245 -24 6-.5 C.

6 EXAMPLE 7 Preparation of 16fi-acetoxyestr-4-en-3-one A solution of mg.of 16fl-hydroxyestr-4-en-3-one in 2 ml. of pyridine and 0.5 ml. ofacetic anhydride is heated on a steam bath for one hour. The hot mixtureis poured into cold, dilute hydrochloric acid, and the steroid isextracted with ether. After washing with dilute hydrochloric acid,water, and sodium bicarbonate solution, the ether extract is dried overmagnesium sulfate, filtered, and concentrated to a residue, whichaffords 75 mg. of 16 ,8- acetoxyestr-4-en-3-one, melting point 87.5-89.5 C. on crystallization from aqueous methanol. This product yields35 mg. of 16fl-acetoxyestr-4-en-3-one, melting point 90.5 0, [M +21.5,

ACHBOH 238 m 615,000

IBEX- after two recrystallizations from aqueous acetone. Additionalproduct is available in the mother liquors.

EXAMPLE 8 Preparation of 16a-acetoxyestr-4-en-3-one A solution of mg. ofl6a-hyclroxyestr-4-en-3-one in 5 ml. of pyridine and 1 ml. of aceticanhydride is allowed to stand overnight at room temperature. Thereaction mixture is poured into water, and the aqueous mixture isextracted with methylene chloride. The methylene chloride extract iswashed with water, dried over magnesium sulfate, filtered, and themethylene chloride is evaporated. The residue is chromatographed on acolumn composed of 50 g. of diatomaceous earth moistened with 25 ml. ofthe lower phase of an ethylene glycol monomethyl ether-heptane (1:1)system. Elution of the column with the upper phase of the above solventsystem affords the 1*6a-acetoxyestr-4-en-3-one at about 2.6 columnretention volumes. The acetate, a noncrystalline oil with no opticalrotation (:4),

xCHaOH 239.5 mp, 615,000

max.

is isolated from the eluate by evaporation of the solvent under reducedpressure.

We claim:

1. The method of preparing 16fl-hydroxyestr-4-en-3- one which comprisescontacting Cephalosporium acremonium in a fermentation media withestr-4en-3-one and recovering the compound therefrom.

2. The method of preparing estr-4-ene-3,16-dione which comprisescontacting Cephalosporium acremonium with estr-4en-3-one underfermentation conditions separating the product resulting and recoveringthe compound therefrom.

3. A method of preparing a member of the group consisting of 16-hydroxyestr-4-en-3-one and 16-ketoestr-4- en-3-one which comprises fermentingestr-4-en-3-one with a growing culture of Bacillus megaterium andrecovering the compounds therefrom.

4-. The method of preparing 163-hydroxyestr-4-en-3- one which comprisesfermenting estr-4-en-3-one with a growing culture of Bacillus megateriumand recovering the compound therefrom.

References Cited UNITED STATES PATENTS 3,188,325 6/1965 Amici et all9551 X A. LOUIS MONACELL, Primary Examiner.

ALVIN E. TANENHOLTZ, Examiner.

1. THE METHOD OF PREPARING 16B-HYDROXYESTR-4-EN-3ONE WHICH COMPRISESCONTACTING CEPHALOSPORIUM ACREMONIUM IN A FERMENTATION MEDIA WITHESTR-4-EN-3-ONE AND RECOVERING THE COMPOUND THEREFORM.
 2. THE METHOD OFPREPARING ESTR-4-ENE-3,16-DIONE WHICH COMPRISES CONTACTINGCEPHALOSPORIUM ACREMONIUM WITH ESTR-4-EN-3-ONE UNDER FERMENTATIONCONDITIONS SEPARATING THE PRODUCT RESULTING AND RECOVERING THE COMPOUNDTHEREFROM.
 3. THE METHOD OF PREPARING A MEMBER OF THE GROUP CONSISTINGOF 16-HYDROXY ESTR-4-EN-O-ONE AND 16-KETOESTR-4EN-3-ONE WHICH COMPRISESFERMENTING ESTR-4-EN-3-ONE WITH A GROWING CULTURE OF BACILLUS MEGATERIUMAND RECOVERING THE COMPOUNDS THEREFROM.